The transporter associated with antigen processing complex comprises three subunits, TAP1, TAP2, and tapasin. Of these, the TAP1 and TAP2 subunits belong to the ATP binding cassette (ABC) family of transmembrane transporters, and function to translocate peptides across the endoplasmic reticulum (ER) membrane. Tapasin interacts with both TAP subunits, and is required for enhancing the structural stability of the TAP1/TAP2 complex. The ER-luminal domain of tapasin also interacts with major histocompatibility complex (MHC) class I molecules, facilitating their assembly. In the proposed studies, we first focus on the molecular events in the cytosol, and will investigate the functions of TAP1 and TAP2 nucleotide binding domains (NBD) during peptide translocation by TAP complexes. We examine evidence for the existence of conformations of the TAP NBD that resemble NBD interactions in the DNA repair enzyme Rad50. In such an interaction, each TAP nucleotide binding site would be comprised of residues from the Walker A motif of one NBD and the signature motif of the second NBD. Using viral inhibitors of TAP, TAP substrates, and appropriately designed TAP1 and TAP2 mutants expressed in insect cells, we will attempt to reconstruct the sequence of molecular events that occur during a TAP catalytic cycle. In the second part of the proposed studies, the focus is upon the ER-luminal face of TAP complexes, in particular on the dynamics of peptide/MHC class I and tapasin/MHC class I interactions. We have been able to demonstrate direct binding between various peptide-deficient MHC class I molecules and tapasin. In the proposed studies, we investigate a competitive displacement model for tapasin function, in which peptides and tapasin compete for class I heavy chain binding. We will examine which domains of tapasin and MHC class I are important for complex formation. We also examine the hypotheses that the tapasin-dependence vs. independence of MHC class I molecules results from intrinsic properties of the class I molecule. We seek to understand the functional consequences of enhanced TAP/tapasin binding in the ER by particular class I molecules. Taken together, these studies will provide insights into how peptide transport into the ER, and peptide assembly with MHC class I molecules, are orchestrated within the cell ? ?
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